Formaldehyde is common byproduct formed in the oxidative demethylation of proteins, nucleic acids, and biological small molecules
Conventional methods for measuring enzyme-mediated oxidative demethylation are limited and cannot be used in high throughput screening for modulators of the enzymes. For example, known assays for formaldehyde formation from a demethylase reaction involve the use of radiolabeled (14C or 3H) methyl-peptide substrates with the subsequent formation, isolation and quantitation of radioactive formaldehyde. Alternative methods have included measurement of the formation of hydrogen peroxide as a byproduct of enzyme activity or Western blotting using methyl lysine specific antibodies. The measurement of hydrogen peroxide product to determine the activity of oxidative demethylases can be problematic in drug screening scenarios since the peroxide formed can react with the test compound or with other components of the demethylase reaction.
Despite their biological importance, oxidative demethylase enzyme activities have proven difficult to quantitatively assay hindering understanding of their kinetic properties, substrate specificities, and reaction mechanisms.
Thus, there is a continuing need for methods and compositions for detection of formaldehyde byproducts of enzyme activity and sensitive assays for enzyme activity.